The nuclear transport factor CSE1 drives macronuclear volume increase and macronuclear node coalescence in Stentor coeruleus.

Stentor coeruleus provides a unique opportunity to study how cells regulate nuclear shape because its macronucleus undergoes a rapid, dramatic, and developmentally regulated shape change. We found that the volume of the macronucleus increases during coalescence, suggesting an inflation-based mechanism. When the nuclear transport factor, CSE1, is knocked down by RNAi, the shape and volume changes of the macronucleus are attenuated, and nuclear morphology is altered. CSE1 protein undergoes a dynamic relocalization correlated with nuclear shape changes, being mainly cytoplasmic prior to nuclear coalescence, and accumulating inside the macronucleus during coalescence. At the end of regeneration, CSE1 protein levels are reduced as the macronucleus returns to its pre-coalescence volume. We propose a model in which nuclear transport via CSE1 is required to increase the volume of the macronucleus, thereby decreasing the surface-to-volume ratio and driving coalescence of the nodes into a single mass.

Developmental exposure to domoic acid targets reticulospinal neurons and leads to aberrant myelination in the spinal cord.

Harmful algal blooms (HABs) produce neurotoxins that affect human health. Developmental exposure of zebrafish embryos to the HAB toxin domoic acid (DomA) causes myelin defects, loss of reticulospinal neurons, and behavioral deficits. However, it is unclear whether DomA primarily targets myelin sheaths, leading to the loss of reticulospinal neurons, or reticulospinal neurons, causing myelin defects. Here, we show that while exposure to DomA at 2 dpf did not reduce the number of oligodendrocyte precursors prior to myelination, it led to fewer myelinating oligodendrocytes that produced shorter myelin sheaths and aberrantly wrapped neuron cell bodies. DomA-exposed larvae lacked Mauthner neurons prior to the onset of myelination, suggesting that axonal loss is not secondary to myelin defects. The loss of the axonal targets may have led oligodendrocytes to inappropriately myelinate neuronal cell bodies. Consistent with this, GANT61, a GLI1/2 inhibitor that reduces oligodendrocyte number, caused a reduction in aberrantly myelinated neuron cell bodies in DomA-exposed fish. Together, these results suggest that DomA initially alters reticulospinal neurons and the loss of axons causes aberrant myelination of nearby cell bodies. The identification of initial targets and perturbed cellular processes provides a mechanistic understanding of how DomA alters neurodevelopment, leading to structural and behavioral phenotypes.

Placozoan fiber cells: mediators of innate immunity and participants in wound healing.

Placozoa is a phylum of non-bilaterian marine animals. These small, flat organisms adhere to the substrate via their densely ciliated ventral epithelium, which mediates mucociliary locomotion and nutrient uptake. They have only six morphological cell types, including one, fiber cells, for which functional data is lacking. Fiber cells are non-epithelial cells with multiple processes. We used electron and light microscopic approaches to unravel the roles of fiber cells in Trichoplax adhaerens, a representative member of the phylum. Three-dimensional reconstructions of serial sections of Trichoplax showed that each fiber cell is in contact with several other cells. Examination of fiber cells in thin sections and observations of live dissociated fiber cells demonstrated that they phagocytose cell debris and bacteria. In situ hybridization confirmed that fiber cells express genes involved in phagocytic activity. Fiber cells also are involved in wound healing as evidenced from microsurgery experiments. Based on these observations we conclude that fiber cells are multi-purpose macrophage-like cells. Macrophage-like cells have been described in Porifera, Ctenophora, and Cnidaria and are widespread among Bilateria, but our study is the first to show that Placozoa possesses this cell type. The phylogenetic distribution of macrophage-like cells suggests that they appeared early in metazoan evolution.

α-Synuclein-112 Impairs Synaptic Vesicle Recycling Consistent With Its Enhanced Membrane Binding Properties.

Synucleinopathies are neurological disorders associated with α-synuclein overexpression and aggregation. While it is well-established that overexpression of wild type α-synuclein (α-syn-140) leads to cellular toxicity and neurodegeneration, much less is known about other naturally occurring α-synuclein splice isoforms. In this study we provide the first detailed examination of the synaptic effects caused by one of these splice isoforms, α-synuclein-112 (α-syn-112). α-Syn-112 is produced by an in-frame excision of exon 5, resulting in deletion of amino acids 103-130 in the C-terminal region. α-Syn-112 is upregulated in the substantia nigra, frontal cortex, and cerebellum of parkinsonian brains and higher expression levels are correlated with susceptibility to Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA). We report here that α-syn-112 binds strongly to anionic phospholipids when presented in highly curved liposomes, similar to α-syn-140. However, α-syn-112 bound significantly stronger to all phospholipids tested, including the phosphoinositides. α-Syn-112 also dimerized and trimerized on isolated synaptic membranes, while α-syn-140 remained largely monomeric. When introduced acutely to lamprey synapses, α-syn-112 robustly inhibited synaptic vesicle recycling. Interestingly, α-syn-112 produced effects on the plasma membrane and clathrin-mediated synaptic vesicle endocytosis that were phenotypically intermediate between those caused by monomeric and dimeric α-syn-140. These findings indicate that α-syn-112 exhibits enhanced phospholipid binding and oligomerization in vitro and consequently interferes with synaptic vesicle recycling in vivo in ways that are consistent with its biochemical properties. This study provides additional evidence suggesting that impaired vesicle endocytosis is a cellular target of excess α-synuclein and advances our understanding of potential mechanisms underlying disease pathogenesis in the synucleinopathies.

The ventral epithelium of Trichoplax adhaerens deploys in distinct patterns cells that secrete digestive enzymes, mucus or diverse neuropeptides.

The disk-shaped millimeter-sized marine animal, Trichoplax adhaerens, is notable because of its small number of cell types and primitive mode of feeding. It glides on substrates propelled by beating cilia on its lower surface and periodically pauses to feed on underlying microorganisms, which it digests externally. Here, a combination of advanced electron and light microscopic techniques are used to take a closer look at its secretory cell types and their roles in locomotion and feeding. We identify digestive enzymes in lipophils, a cell type implicated in external digestion and distributed uniformly throughout the ventral epithelium except for a narrow zone near its edge. We find three morphologically distinct types of gland cell. The most prevalent contains and secretes mucus, which is shown to be involved in adhesion and gliding. Half of the mucocytes are arrayed in a tight row around the edge of the ventral epithelium while the rest are scattered further inside, in the region containing lipophils. The secretory granules in mucocytes at the edge label with an antibody against a neuropeptide that was reported to arrest ciliary beating during feeding. A second type of gland cell is arrayed in a narrow row just inside the row of mucocytes while a third is located more centrally. Our maps of the positions of the structurally distinct secretory cell types provide a foundation for further characterization of the multiple peptidergic cell types in Trichoplax and the microscopic techniques we introduce provide tools for carrying out these studies.

Cells containing aragonite crystals mediate responses to gravity in Trichoplax adhaerens (Placozoa), an animal lacking neurons and synapses.

Trichoplax adhaerens has only six cell types. The function as well as the structure of crystal cells, the least numerous cell type, presented an enigma. Crystal cells are arrayed around the perimeter of the animal and each contains a birefringent crystal. Crystal cells resemble lithocytes in other animals so we looked for evidence they are gravity sensors. Confocal microscopy showed that their cup-shaped nuclei are oriented toward the edge of the animal, and that the crystal shifts downward under the influence of gravity. Some animals spontaneously lack crystal cells and these animals behaved differently upon being tilted vertically than animals with a typical number of crystal cells. EM revealed crystal cell contacts with fiber cells and epithelial cells but these contacts lacked features of synapses. EM spectroscopic analyses showed that crystals consist of the aragonite form of calcium carbonate. We thus provide behavioral evidence that Trichoplax are able to sense gravity, and that crystal cells are likely to be their gravity receptors. Moreover, because placozoans are thought to have evolved during Ediacaran or Cryogenian eras associated with aragonite seas, and their crystals are made of aragonite, they may have acquired gravity sensors during this early era.

Phaeocystis antarctica blooms strongly influence bacterial community structures in the Amundsen Sea polynya.

Rising temperatures and changing winds drive the expansion of the highly productive polynyas (open water areas surrounded by sea ice) abutting the Antarctic continent. Phytoplankton blooms in polynyas are often dominated by the haptophyte Phaeocystis antarctica, and they generate the organic carbon that enters the resident microbial food web. Yet, little is known about how Phaeocystis blooms shape bacterial community structures and carbon fluxes in these systems. We identified the bacterial communities that accompanied a Phaeocystis bloom in the Amundsen Sea polynya during the austral summers of 2007-2008 and 2010-2011. These communities are distinct from those determined for the Antarctic Circumpolar Current (ACC) and off the Palmer Peninsula. Diversity patterns for most microbial taxa in the Amundsen Sea depended on location (e.g., waters abutting the pack ice near the shelf break and at the edge of the Dotson glacier) and depth, reflecting different niche adaptations within the confines of this isolated ecosystem. Inside the polynya, P. antarctica coexisted with the bacterial taxa Polaribacter sensu lato, a cryptic Oceanospirillum, SAR92 and Pelagibacter. These taxa were dominated by a single oligotype (genotypes partitioned by Shannon entropy analysis) and together contributed up to 73% of the bacterial community. Size fractionation of the bacterial community [<3 µm (free-living bacteria) vs. >3 µm (particle-associated bacteria)] identified several taxa (especially SAR92) that were preferentially associated with Phaeocystis colonies, indicative of a distinct role in Phaeocystis bloom ecology. In contrast, particle-associated bacteria at 250 m depth were enriched in Colwellia and members of the Cryomorphaceae suggesting that they play important roles in the decay of Phaeocystis blooms.

Neurotransmitter modulation of extracellular H+ fluxes from isolated retinal horizontal cells of the skate.

Self-referencing H(+)-selective microelectrodes were used to measure extracellular H(+) fluxes from horizontal cells isolated from the skate retina. A standing H(+) flux was detected from quiescent cells, indicating a higher concentration of free hydrogen ions near the extracellular surface of the cell as compared to the surrounding solution. The standing H(+) flux was reduced by removal of extracellular sodium or application of 5-(N-ethyl-N-isopropyl) amiloride (EIPA), suggesting activity of a Na(+)-H(+) exchanger. Glutamate decreased H(+) flux, lowering the concentration of free hydrogen ions around the cell. AMPA/kainate receptor agonists mimicked the response, and the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) eliminated the effects of glutamate and kainate. Metabotropic glutamate agonists were without effect. Glutamate-induced alterations in H(+) flux required extracellular calcium, and were abolished when cells were bathed in an alkaline Ringer solution. Increasing intracellular calcium by photolysis of the caged calcium compound NP-EGTA also altered extracellular H(+) flux. Immunocytochemical localization of the plasmalemma Ca(2+)-H(+)-ATPase (PMCA pump) revealed intense labelling within the outer plexiform layer and on isolated horizontal cells. Our results suggest that glutamate modulation of H(+) flux arises from calcium entry into cells with subsequent activation of the plasmalemma Ca(2+)-H(+)-ATPase. These neurotransmitter-induced changes in extracellular pH have the potential to play a modulatory role in synaptic processing in the outer retina. However, our findings argue against the hypothesis that hydrogen ions released by horizontal cells normally act as the inhibitory feedback neurotransmitter onto photoreceptor synaptic terminals to create the surround portion of the centre-surround receptive fields of retinal neurones.

Mitochondrial respiration and Ca2+ waves are linked during fertilization and meiosis completion.

Fertilization increases both cytosolic Ca(2+) concentration and oxygen consumption in the egg but the relationship between these two phenomena remains largely obscure. We have measured mitochondrial oxygen consumption and the mitochondrial NADH concentration on single ascidian eggs and found that they increase in phase with each series of meiotic Ca(2+) waves emitted by two pacemakers (PM1 and PM2). Oxygen consumption also increases in response to Ins(1,4,5)P(3)-induced Ca(2+) transients. Using mitochondrial inhibitors we show that active mitochondria sequester cytosolic Ca(2+) during sperm-triggered Ca(2+) waves and that they are strictly necessary for triggering and sustaining the activity of the meiotic Ca(2+) wave pacemaker PM2. Strikingly, the activity of the Ca(2+) wave pacemaker PM2 can be restored or stimulated by flash photolysis of caged ATP. Taken together our observations provide the first evidence that, in addition to buffering cytosolic Ca(2+), the egg's mitochondria are stimulated by Ins(1,4,5)P(3)-mediated Ca(2+) signals. In turn, mitochondrial ATP production is required to sustain the activity of the meiotic Ca(2+) wave pacemaker PM2.